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4 OCTOBE R 2017

Scientific Background on the Nobel Prize in Chemistry 2017

T H E D E V E L O P M E N T O F C RYO - E L E C T RO N M I C RO S C O P Y

THE ROYAL SWEDISH ACADEMY OF SCIENCES has as its aim to promote the sciences and strengthen their influence in society.
BOX 50005 (LILLA FRESCATIVÄGEN 4 A), SE-104 05 STOCKHOLM, SWEDENTEL +46 8 673 95 00, KVA@KVA.SE  WWW.KVA.SENobel Prize ® and the Nobel Prize ® medal design markare registrated trademarks of the Nobel Foundation

The Royal Swedish Academy of Sciences has decided to award Jacques Dubochet, Joachim
Frank and Richard Henderson the Nobel Prize for Chemistry 2017 for "developing cryoelectron microscopy for the high-resolution structure determination of biomolecules insolution".

Introduction
In 1968, George Gamow, a physicist, and Martynas Yčas, a microbiologist, published a popularscience book, Mr. Tompkins Inside Himself: Adventures in the New Biology, which tells a storyabout Mr. Tompkins as he explores the cellular architecture of his body on a dream journey
through his bloodstream, guided by his doctor. While inspecting the structural details of single
cells and organelles, Mr. Tompkins' guide enthusiastically informs him that this knowledge isbased on studies using the electron microscope.
Mr. Tompkins' tour makes it obvious that at that time the instrument had already broughtstudies of biological material to a previously unimaginable level of detail. However, until just afew years ago scientists could still only dream of being able to use the electron microscope tozoom in further into cells and organelles, in order to uncover the atomic details of thebiomolecules that underpin their architecture and function.
This dream became reality recently when a series of critical developments made it possible totake full advantage of the pioneering discoveries and improvements made by Jacques Dubochet,
Joachim Frank and Richard Henderson. These advances now allow structural determination of
non-crystalline biomolecules in solution at high resolution, using single-particle 1 cryo-electron
microscopy (EM).

Challenges in structural studies of biological material
Short after the experimental demonstration of an electron microscope by Ernst Ruska, for which
he was honoured with the Nobel Prize for Physics in 1986 (1), Ladislaus Marton published apaper (2) that commented on Ruska's discovery. In this short report, Marton noted that the new
instrument unfortunately could not be used to study biological material without the "destructionof the organic cells by the intense electronic bombardment".
Preventing such destruction would require a new sample-preparation technique. Marton
proposed visionary solutions to the problem: cooling the biological material or the use of an
approach similar to negative staining. Another major problem was how to preserve water in thebiological sample in the vacuum maintained inside the electron microscope chamber.
And there were even more challenges to face. To mention only the most basic ones, intact
biological material has very low image contrast as most high-energy electrons pass straight
through the specimen. At the same time, the electron dose must be kept low enough to prevent
damage. The probability for multiple electron scattering events must be negligible at the1

The term "particle" is used for biological macromolecules or complexes. The term "single particle" isused to indicate that non-crystalline specimens are analyzed. When using this approach, an ensemble of alarge number of individual particles in solution is analyzed.
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electron energy used; i.e. samples must be thin, ideally comprising a single layer of the particlesof interest. Furthermore, the studied objects often move both upon interacting with electronsand due to drifts in temperature; the movement reduces information content, especially whenusing film or slow detectors to record images. As a result, until recently the resolution wastypically limited to a few nanometres for biological molecules (Fig. 1).

Fig. 1. Models of the electron-transport chain components in a mitochondrial supercomplex I1III2IV1, determinedin 2011 (left, from (3), resolution ~2 nm) and 2016 (right, from (4), resolution ~6 Å), respectively. On the left, thecoloured shape indicates the position of amphipols used for solubilization. On the right, complexes I, III and IV areshown in blue, green and pink, respectively. The encircled inset shows a model of a putative CIII-CIV interface.

Negatively stained biological material
The necessity to use the lowest possible electron intensities to study low-contrast samples
stimulated the development of new sample-preparation methods when recording images ofbiological material. The first commonly and successfully employed method was negative
staining, established in the 1940s and refined during the following 20 years (5-7).
When using this approach, the biological material is embedded in a thin amorphous film of aheavy-metal salt, which generates a cast around the object. The cast scatters electrons morestrongly than the encapsulated material, is more resistant to electron damage, and prevents
collapse of the biological material during drying in the vacuum within the electron microscope.
The approach offered detailed information about the morphology of bacteria, viruses andorganelles. However, for studies of single molecules or molecular complexes, in the best case thepictures could reveal only the envelope of the covered particles with a resolution that is limitedby the granularity of the stain. Nevertheless, the use of this sample-preparation techniqueoffered important low-resolution structural information. The experimental and theoretical tools
used for calculation of three-dimensional (3D) structures from two-dimensional (2D)
projections in the electron microscope established the basis for today's advancements.

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The first high-resolution model of bacteriorhodopsin (31) was based on analysis of millions ofprotein molecules in a 2D crystal, which allowed the spread of the total electron dose over alarge number of particles.
The analysis of a large number of molecules in the 2D crystal is equivalent to averaging directlyin the microscope. For non-periodic assemblies of symmetrical particles, the signal-to-noise
ratio can be increased by averaging over the asymmetrical units. However, for the general caseof non-periodic asymmetrical particles, the challenge was to determine the position andorientation of each particle in an image from weak signals. Once this could be achieved,
averaging would be possible. However, such analyses would require computer power wellbeyond that available in 1990 (36).

Fig. 3. Extract from Table 2 in (37), which addresses thequestion: can single molecule alignment be carried out
in practise? The answer is given to the right for a numberof example proteins, listed to the left, with molecularweights (Da) in the middle column.

Fig. 4. Examples of structures determined using cryo-EM
as of May 2016 [image from (38)]. The figure illustrates
the conclusions from (37). Note that the smallest protein(64 kDa) determined to date using cryo-EM ishaemoglobin (39).

Five years after the publication of the high-resolution structure of bacteriorhodopsin,
Henderson presented a quantitative analysis of the challenges needed to be overcome in order todetermine atomic-resolution structures of non-crystalline molecular assemblies (37). He
concluded that by using low-intensity, non-destructive electron irradiation in phase-contrast
electron microscopy, it would be possible to determine the 2D position and 3D orientation ofindividual particles, given a sufficiently high molecular weight. The information would allow
averaging ensembles of randomly distributed particles, thereby eventually reaching atomicresolution. The conclusion from Henderson's work was that, assuming a molecular weighthigher than ~50 kDa (Fig. 3), it should be possible to align and average a reasonable number(~104) of particles to determine structures at atomic resolution (~3 Å; see Fig. 4). During thecoming years the predicted number of particles necessary to reach this resolution was revised tosmaller numbers (36,40), and Glaeser presented an analysis that suggested that the size limit
could be adjusted to ~20 kDa (36). An analysis along the same lines, but less detailed, was alsopresented in an earlier paper (41).

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Recent and ongoing improvements in image-processing methods and computer programs havealso been essential for the current developments. For example, maximum likelihood algorithms(76,77) became particularly important in electron microscopy when better resolution wasachieved using the new electron detectors.

Summary
The first high-resolution structure, determined using cryo-EM, was presented in 1990. A decadepassed before high-resolution structures of helical and icosahedral particles were imaged,
determined based on analysis of data recorded on film. After the introduction of the new Direct
Electron Detectors in 2012-13 (see Fig. 9) and the first reports of de novo atomic structuralmodels of smaller single particles, such as that of the membrane protein TRPV1 ion channel(78), cryo-EM has very rapidly become a major new tool in structural biology.

Fig. 9. The resolution progression of cryo-EM, illustrated by a representation of glutamate dehydrogenase with an increasing level of detail from left to right. For a protein of this size, 334 kDa, the 1.8 Å
resolution to the right (38) could only be achieved after 2012/13. After an image by V. Falconieri (seeref. 38). Illustration: © Martin Högbom, Stockholm University.

It is captivating to think about the amount of time that has passed before we could get to thispoint. About six decades after John Kendrew's and Max Perutz's pioneering crystallographic
work on myoglobin and haemoglobin (Nobel Prize for Chemistry in 1962 "for their studies of thestructures of globular proteins"), and four decades after the first developments that laid thegroundwork for single-particle cryo-EM, a high-resolution structure of haemoglobin in solution,
determined using cryo-EM, was presented (39).
Single-particle cryo-EM is unique in that it does not require crystallization, uses very small
amounts of material, and covers a wide range of sizes, from particles the size of haemoglobin(64 kDa), to very large particles up to several megadaltons. Cryo-electron tomography is used todetermine structures of even larger objects, including organelles and cells, with the potential of

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